Appropriate implementation of Okazaki fragment maturation during DNA replication in eukaryotic cells is a fundamental mechanism for mutation avoidance and genome stability. During lagging strand DNA synthesis, multiple RNA primers and immediately adjoined DNA-fragments are synthesized by primase (a hetero tetramer of a RNA polymerase and DNA Pol ). However, both enzymes lack a proof reading function. Therefore, this initial RNA-DNA fragment (alpha-segment of the Okazaki fragment) is highly mutagenic and must be processed by nuclease complexes. The parent proposal aims to define detailed molecular mechanism for the nuclease-driven RNA primer processing in eukaryotic nucleus and mitochondrion. For the last funding period, we have defined the roles of several nucleases in these processes, including S. cerevisiae RNase H (35), ScRad27 or human FEN1, and exonuclease-1, and mutagenic consequences when these nucleases are defective. The parent proposal continues our focus to test a central hypothesis that a-segment processing is a vital part of cellular mechanisms to maintain genomic integrity and prevent mutagenic stresses due to intrinsic DNA sequence obstacles and exogenous insults. Deficiency of this integrative machinery could lead to a high incidence of mutagenesis and carcinogenesis. We will further define detailed molecular mechanisms for the nuclease-driven "a-segment" processing in Okazaki fragment maturation during replication of normal DNA sequence and repetitive DNA sequence regions, in the nucleus as well as the mitochondrion. Through a series of vigorous systematic analyses, we intend to obtain a high resolution image of how these nuclease complexes collectively work towards RNA primer processing in different scenarios and to relate in vitro and in vivo data using yeast and mammalian systems, including human cell lines and transgenic mice. Recently, we found that two major nucleases, FEN1 and DNA2, are localized into mitochondria and cooperatively process replication and repair DNA intermediates for ligation and completion of circular mtDNA replication and repair. These novel and exciting observations prompted us to expand our scope: i) to knock out the DNA2 gene to determine if defective DNA2-mediated RNA primer removal causes mitochondrial genomic instabilities and consequently promotes cancers and other genetic diseases and ii) to link functional defects of the DNA2 mutations identified in human mitochondrion-based diseases to pathologic mechanisms. Information made available from these additional studies will establish a relationship among the functions of these novel mitochondrial genes, unique mitochondrial mutagenic phenotype(s), and pathological mechanisms of cancers and other genetic diseases. The proposed study may also set a good foundation for new treatment regimens to patients with mitochondrion-based cancers and other disorders. Moreover, the proposed research will have immediate economic impact by creating 1 postdoctoral position and retaining a graduate student and covering efforts of two key existing scientists. PUBLIC HEALTH RELEVANCE: The proposed studies will establish a relationship among the functions of these novel mitochondrial genes, unique mitochondrial mutagenic phenotype(s), and pathological mechanisms of cancers and other genetic diseases and may provide new treatment regimens to patients with mitochondrion-based cancers and other disorders. Moreover, the proposed research will have immediate economic impact by creating 1 postdoctoral position and retaining a graduate student and covering efforts of two key existing scientists.